All molybdoenzymes examined so far, except nitrogenase, require a molybdenum cofactor for their function. The essentiality of the Mo cofactor for normal human health and development is underlined by the severe pathology and early fatality in the recently discovered human genetic disorder of combined molybdoenzyme deficiency. Such pleiotropy is seen in lower organisms as well, and is most often due to the inability to synthesize the pterin component of the Mo cofactor. In this proposal the ongoing studies on the biosynthesis of Mo cofactors will be continued. Using the chl mutants of Escherichia coli we propose to characterize the enzymes and metabolic intermediates in the de novo pathway of Mo cofactor synthesis. The biosynthesis of the pterin ring, the cascading system for the formation of the dithiolene group, the mechanism of dinucleotide synthesis and the mode of attachment of Mo to the pterin prosthetic group will be examined. Additional studies will probe the role of the pterin component in the catalytic activities of molybdoenzymes. The newly developed technique of electrospray mass spectrometry will be used in an attempt to determine the sizes of protein-bound cofactors. Since it is becoming more and more evident that unequivocal and stereochemical information on the native cofactor can be obtained only from determination of crystal structures, such studies will be initiated on the enzyme DMSO reductase. Recent developments in molecular biology have increased the scope of structure-function studies on enzymes by providing rapid means of preparing defined mutants by site-directed mutagenesis. We have acquired or isolated several DNA clones to supplement the studies described above. Full length clones of nitrate reductase from Arabidopsus thaliana and DMSO reductase from E. coli as well as the partial clone of rat liver sulfite oxidase are available. The latter will be used to prepare a full length clone using polymerase chain reaction or other suitable procedures. The E. coli genomic fragment containing the 5 reading frames of the chlA locus and a plasmid capable of overexpressing the two proteins of the chlEN locus have also been obtained and will be used to facilitate attainments of the goals listed above.